In healthy subjects, blood viscosity and thickness is regulated by a process known as hemostasis. This mechanism prevents loss of blood from the vascular system.
Blood coagulation is regulated by a complex process to stop any bleeding occurring in the body. Stable clots are formed through the interaction of coagulation protein factors, blood vessels and platelets. The process continues after healing, when the blood clot is dissolved.
During the first stages of clot formation, platelets aggregate, at the same time as a phenomenon known as blood cascade is activated. In this process, fibrinogen, a soluble plasma protein, is converted to an insoluble fibrin mesh or blood clot. This conversion is catalysed by thrombin, an enzyme generally present in blood in its inactive form, prothrombin.
Blood disorders arise from imbalances in hemostasis. These can be of a genetic origin, such as in hemophilia or Von Willebrand's disease; triggered by other conditions such as antiphospholipid antibody syndrome, irritable bowel syndrome or cancer; or acquired through extrinsic factors: patients taking oral anticoagulants as treatment or prophylaxis of thrombotic disorders, cardiac or vascular diseases.
Oral anticoagulant therapy, such as warfarin, is widely used and need frequent monitoring because of its narrow therapeutic index. The dosage should be adjusted periodically, in order to avoid thrombosis or risk of bleeding.
For these and other patients with known predisposition conditions such as immobility, obesity, mediation, or undergoing surgery or dental treatment, the availability of reliable tests enabling them to regularly monitor coagulation at their homes would represent a convenient, fast and cheap alternative to the clinic coagulation tests currently available. Such tests may also be employed as a preliminary aid in the diagnosis of hemostatic disorders.
The world's most common coagulation analysis is the so-called International Normalised Ratio (INR). This ratio is calculated through the Prothrombin Time (PT), which is the time elapsed from activation by the coagulating agent to the start of blood clotting. The activation agent is a tissue factor or thromboplastin and this mechanism is called the “extrinsic” pathway. Because of differences between different batches and manufacturers of tissue factor (it is a biologically obtained product), the INR was devised to standardise the results. The INR is the ratio of a patient's prothrombine time to the mean prothrombin time (MNPT) of at least 20 healthy normal people, raised to the power of the international Sensitivity Index (ISI) value for the control sample used. Each manufacturer gives an ISI for any factor tissue commercialised, indicating how the particular batch of tissue factor compares to an internationally standardized sample.
There is a second, but less commonly used analysis type, which consists of an analogous coagulation mechanism, through the “intrinsic” pathway, and it is called the Activated Partial Prothrombin Time (APTT). Both of these analyses are referred to as clotting times in the present application.
Traditionally, in Europe, these analyses were carried out in laboratories, where blood sample preparation is usually required prior to determining the PT. In recent years an emerging trend to employ Point-of-Care (POC) devices, or similarly named Nearly-Patient-Testing (NPT), to be used directly by the nurse or physician, or autonomously by the patient, has taken place and has largely replaced traditional methods.
The methods that were developed initially and known in the art required extraction of large or exact volumes of blood by venipuncture, subsequent treatment of blood prior to running the test and expert personnel to perform the process and interpret the results. In contrast, Point-of-care coagulometers, also known as portable coagulometers, require a whole blood droplet extracted by fingerpricking and provide immediate INR results.
Patent application WO 92/21028 describes a detection method based on ferromagnetism. The device contains a coagulation chamber and a control chamber, each of which is fitted with an agitating vane, which rotates in an oscillating magnetic field. The rotation of the vane in the coagulation chamber slows down as the coagulation of blood starts and exerts resistance against its movement. The coagulation time is measured as the time at which the relative movement of the agitation vanes in the chambers changes.
Other devices, such as those in U.S. Pat. No. 5,110,727 contain a blood sample with metallic particles dispersed through it. When an oscillating magnetic field is applied, a back and forth movement of the particles is induced that slows down as blood coagulates. The decrease in speed correlates to the increase of blood sample viscosity or the start of coagulation.
Patent application WO 00/06761 and WO 02/48707 A2 describe both a device fitted with electrodes in contact with a stationary blood sample and measure, respectively, the variation in electrical conductivity and current as blood viscosity increases.
WO 2004/059316 A1 describes a low cost, disposable device for determining clotting time of blood. The device is fitted with a microsensor, at least partially in contact with the fluid and measures the impedance and capacitance of the blood in the channel when blood coagulates and the flow stops.
However, high production costs associated with these devices restrict their use as disposable units.
Therefore, there remained a need for accurate, low cost disposable chips and detection methods for POC and/or NPT clotting time determination.
There has been a development towards detection tests of smaller size, requiring smaller and unmeasured whole blood samples, in the microliter scale, due to the advances in materials science and in electronic and optical methods.
Patent Application WO 2007/025559 A1 discloses a multi-layer device for the determination of coagulation in a plasma or whole blood sample, comprising one or more detection areas, all of them provided with at least one coagulation stimulation reagent.
Patent application US2007/0122849A1 discloses a sample assay structure in a microfluidic chip for quantitative analysis and detection of analytes.
EP 0394070 B1 describes a microfluidic device of one capillary channel, optimised for determining the APTT in a whole blood sample, of 40 μL of volume and residence time of 200 s. The device uses as reagent a mixture of an activated agent for activated partial thromboplastin time measurements and a mixture of phospholipids. The detection method employed through the capillary track is visual or optical, such as a LED, and determines the APTT when the blood flow stops along the device.
U.S. Pat. No. 6,900,021 describes a microfluidic device to conduct in vitro studies on the reaction and effects of various compounds on cells. The fluid flow is controlled using pumps, pressure differences or electrical fields, and not by capillarity in the microfluidic channel. There are two inlet flow paths intersecting and merging with a main flow path to allow the reaction to occur. Therefore, the main flow path does not comprise an area containing a reagent. Further, the reagents are not present in the chip, but added at different points and times, this allows the chip to be used for different reaction assays with different reagents.
Despite these developments, the point of care coagulometers being used today still have important drawbacks:                although most of the chips or test strips used are disposable, they include several components such as means to collect the blood sample, means to measure the change in conductivity or means for measuring the change in viscosity. The presence of active components such as electrochemical contacts or oscillating particles in the strip makes the production of the disposable chip complex and expensive. Further, the size cannot be reduced without compromising the quality of the strip.        Although advances have been made concerning the amount of blood sample needed for the test, the volume is still in the range of 10 μl in the best of cases, which is still inconvenient for the patient. This compares unfavourably, for example, with the amount used for other tests such as glucose measuring, which can be accurately done with a sample of blood of 1 μl or less.        The detection and measuring apparatus that are used with the known test strips or chips are still rather complex. In some cases they need additional means to convey or move the blood sample, such as magnetic fields or pumps. In others the device needs several detection means: electrochemical or magnetic means to measure some property changes in the sample that require calibration chips, and additional detection means to read additional on-board quality control systems. This increases the complexity and therefore the cost of the portable device.        
In view of these drawbacks, it is an object of the present invention to provide an improved microfluidic device and method for determining clotting time in a fluid medium such as blood or plasma, which involves only minimal steps, has a low cost, and can thus be used autonomously by the patient. It is another object to provide a measuring device to be used with the microfluidic device, such as a coagulometer, in order to detect and monitor the clotting time of the sample and the quality controls present in the microfluidic device, which is simple to manufacture, is compact and can be autonomously used by the patient.